Agent for relieving side effects caused by immunosuppressants

ABSTRACT

The present invention relates to an agent for relieving side effects caused by immunosuppressants, which comprises HGF (Hepatocyte growth factor) as an active component, a method for relieving side effects caused by immunosuppressants, which comprises administration of HGF and use of HGF for producing an agent for relieving the side effects. HGF as an active component can reduce multiple-organ or systemic side effects caused by immunosuppressants. Therefore, according to the present invention, restrictions of use and dose of immunosuppressants are reduced, success rate of organ transplantation and cure rate of various patients to which the immunosuppressants are administered can be improved and at the same time burden of the patients can be remarkably reduced.

TECHNICAL FIELD

The present invention relates to an agent for relieving side effectscaused by immunosuppressants. More particularly, the present inventionrelates to an agent for relieving side effects which can ease sideeffects caused by immunosuppressants and which contains HGF (HepatocyteGrowth Factor) as an effective component.

BACKGROUND ART

An immunosuppressant is conventionally used for treatment of anautoimmune disease and malignant tumor, suppression of graft rejectionreaction in organ transplantation and the like. An immunosuppressant isa medicine which suppresses an excessively occurring immune response,and is largely classified into a non-specific immunosuppressant or aspecific immunosuppressant depending on its specificity ofimmunosuppression.

Examples of the non-specific immunosuppressant include an adrenocorticalhormone (for example, cortisone, dexamethasone or the like),antimetabolic (for example, 6-mercaptopurine, azathiopurine,5-fluorouracil, methotrexate or the like), alkylating agent (forexample, cyclophosphamide, busulfan or the like), alkaloid (for example,vinblastine, vincristine or the like), antibiotic (for example,cyclosporin A and G, FK506 (tacrolimus), mitomycin C, daunorubicin orthe like), and the like. Examples of the specific immunosuppressantinclude an anti-lymphocyte globulin, monoclonal antibodies (for example,anti-CD3 antibody, anti-CD4 antibody and the like) against variousantigens expressed on the surface of a lymphocyte, and the like.

Among the above-described immunosuppressants, cyclosporin and FK506 havestrong immunosuppression action and accomplish excellent results insuppression of graft rejection reaction in organ transplantation of akidney, liver, heart, pancreas and the like, therefore, are notedmedicine. Both of cyclosporin and FK506 are antibioticimmunosuppressants having a cyclic peptide structure, and is believed toexpress immunosuppressant effect by blocking an intracellular signaltransfer path which introduces expression of a cytokine gene such asIL-2 or the like via activation of a T-cell antigen receptor (TCR) dueto antigen stimulation in a T-cell system. More specifically, each oneof cyclosporin and FK506 is known to be bound in a cell to a bindingprotein thereof, and cyclosporin is bound to cyclophilin and FK506 isbound to an FK506 binding protein. These binding proteins are calledimmunophilin, including their families. The following study revealedthat a complex of immunophilin with FK506 or cyclosporin is bound to aserine/threonine dephosphorylation enzyme, calcineurin, and inhibits itsenzymatic activity. And now, cyclosporin and FK506 are believed toexpress immunosuppressant action since a complex of immunophilin withthem inhibits enzymatic activity of calcineurin. This theory issupported by the fact that there is a correlation between strength ofinhibition of enzymatic activity of calcineurin by a derivative ofcyclosporin and FK506 and strength of inhibition of IL-2 synthesis intheir T-cells, or the like.

As described above, a lot of immunosuppressants are used, however, anymedicine has side effects. For example, multiple-organ or systemic sideeffects have been reported, such as bone marrow suppression, leukopenia,thrombocytopenia, severe infectious disease, hepatic disorder, kidneydisorder, neuropathy, lung disorder, gastrointestinal disorder,anorexia, nausea, vomiting, diarrhea, anemia, gingivostomatitis,alopecia, piloerection, chromatosis, hypotension, arrhythmia, fever,convulsion, infecundity, azoospermia, malaise and the like. Thus,immunosuppressants now used have problems that side effect is strong,dose thereof is restricted and careful attention is necessary in use.

More particularly, an immunosuppressant having strong bone marrowsuppression causes leukopenia and easily manifests severe infectiousdisease, therefore, the dose thereof is necessary to be restricted.Then, acute rejection reaction easily to be manifested in organtransplantation, and dysfunction of transplanted organs often occurs.Cyclosporin and FK506 generally used in organ transplantation aremedicines which suppress IL-2 synthesis by acting T-cells since they canremarkably reduce critical rate of acute rejection reaction after organtransplantation. Therefore, cyclosporin and FK506 have advantages thatmanifesting rate of bone marrow suppression is low, manifestation ofsevere infectious disease following leukopenia can be protected, andmanagement after organ transplantation will be easier. Also due to theseeffects, the result of organ transplantation can be remarkably improved.

Further, even for cyclosporin and FK506, side effects are observed. Forexample, for cyclosporin, multiple-organ or systemic side effects suchas nephrotoxicity, hepatoxicity, neuropathy, hypertension, caput femorisnecrosis, cataract, diabetes, acute pancreatitis, cytomegalovirusinfectious disease and the like have been reported, and the dose thereofis restricted. Especially in renal transplantation, kidney disorderbased on nephrotoxicity has serious influence on aftercare of thetransplanted kidney, and further in early period after thetransplantation, the kidney disorder based on nephrotoxicity isdifficult to be distinguished from renal insufficiency due to acuterejection reaction after the transplantation. Therefore, it isindispensable to monitor constantly cyclosporin concentration in bloodand carefully control the dose, and if side effects by cyclosporin arerecognized, the dose of cyclosporin should be reduced or theadministration should be stopped, and after transplantation managementagainst side effects is necessary. Further, the dose of FK506 can bereduced since FK506 is 100 times as active as cyclosporin, thereforeside effects caused by FK506 can be reduced as compared withcyclosporin. However, as the side effects caused by FK506,multiple-organ or systemic side effects, for example, kidney disorder,anorexia, vomiting, pancreatitis, hyperkalemia, hyperuricemia and thelike have been reported.

It is also reported that calcium-channel blocker and prostaglandin areuseful in prevention of nephrotoxicity caused by cyclosporin(Transplantation 51, 293-295, 1991; Clin. Nephrol 25 suppl. 1!, S89-S94,1986). However, these medicines have insufficient effect and can notsuppress multiple-organ or systemic side effects.

As described above, immunosuppressants now used have severemultiple-organ or systemic side effects, therefore, there are manyrestrictions with respect to use and dose and a medicine is intenselyrequired which can remarkably reduce the multiple-organ or systemic sideeffects of the immunosuppressants.

The present inventor has studied intensely on medicine which can reducethe side effects of the immunosuppressants to resolve theabove-mentioned problems. As the result, the inventor has found that HGFcan remarkably reduce the multiple-organ or systemic side effects of theimmunosuppressants.

More specifically, HGF is a protein that the present inventor et al.have found as a factor which enhances proliferation of mature liverparenchyma cells in vitro and purified from serum of rats withregenerating liver (Biochem Biophys Res Commun, 122, 1450, 1984).Further, the inventor et al. have succeeded in isolation of HGF from ratblood platelets (Proc. Natl. Acad. Sci, 83, 6489, 1986; FEBS Letters,22, 311, 1987), and have determined partial amino acid sequence thereof.Further, the present inventor et al. have succeeded in obtaining HGF asa protein by cloning HGF cDNA of human and rat origin utilizing thesolved HGF amino acid sequence, and introducing the cDNA into animalcells by recombinant technique (human HGF: Nature, 342, 440, 1989; ratHGF: Proc. Natl. Acad. Sci, 87, 3200, 1990).

This HGF found as a factor which specifically proliferates liverparenchyma cells has been proved, by recent studies of a lot ofresearchers including the present inventor, to manifest variousphysiological activities in vivo and to be acting on therapy of injuryof various organs and tissues. HGF is expected to be not only a researchobject but also a medicine applied to human or animal therapeuticagents.

In view of such action of HGF, the present inventor has thought that HGFmust be useful for reduction of side effects caused byimmunosuppressants and has studied the effect of HGF against sideeffects of immunosuppressants. As the result, it has been found that HGFcan remarkably reduce multiple-organ or systemic side effects caused byimmunosuppressants. It is a novel knowledge and an action of HGF whichhas conventionally not been known that HGF is effective in reduction ofside effects caused by immunosuppressants.

The present invention has been accomplished based on such knowledge, andan object of the present invention is to provide an agent for relievingside effects caused by immunosuppressants.

DISCLOSURE OF THE INVENTION

The present invention is an agent for relieving side effects caused byimmunosuppressants, which comprises HGF as an active component.

Other inventions of the present invention are a method for relievingside effects caused by immunosuppressants, which comprisesadministration of an effective amount of HGF and use of HGF forproducing an agent for relieving side effects caused byimmunosuppressants.

The above-mentioned HGF may be one derived from human or animal tissueor blood, or may be one produced by gene engineering.

Because cells on which HGF as an effective component acts exist inextremely wide range of organs and tissues, multiple-organ or systemicside effects caused by immunosuppressants can be reduced.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph which shows experiment design and survival rate ofmice.

BEST MODES OF CARRYING OUT THE INVENTION

As to HGF which is the active ingredient of the invention, any HGF canbe used in the invention as long as it is purified to be able to use fora medicine, regardless of preparation methods of HGF. Many methods areknown to prepare HGF, and, for example, HGF can be obtained byextraction and purification from organs such as liver, spleen, lung,bone marrow, brain, kidney, placenta and the like, blood cells such asplatelets, leukocytes and the like, plasma and serum of mammals such asrat, cow, horse, sheep and the like. Also, it is possible to obtain HGFby cultivation of primary culture cells or cell lines producing HGF,followed by separation and purification from the culture product (e.g.culture supernatant, cultured cell, etc.). Further, HGF can be obtainedby gene engineering method which comprises cloning the gene coding HGFwith a proper vector, inserting it into a proper host cell to give atransformant, and separating the desired recombinant HGF from theculture supernatant of the transformant (e.g. Nature, 342, 440, 1989,Japanese Patent Kokai No. 111383/1993, Biochem. Biophys. Res. Commun.,163, 967, 1989). The host cell is not specifically limited, and varioushost cells conventionally used in gene engineering methods can be used,which are, for example, Escherichia coli, Bacillus subtilis, yeast,filamentous fungi, and plant or animal cells.

More specifically, the method of extracting and purifying HGF from livetissues is, for example, to administer carbon tetrachloride to a ratintraperitoneally, remove a liver from the rat with hepatitis, grind it,and purify by the ordinary protein purifying technique such as gelcolumn chromatography using S-Sepharose and heparin Sepharose, HPLC andthe like. Further, by the gene engineering method, the gene coding theamino acid sequence of human HGF is cloned into a vector such as bovinepapilloma virus DNA and the like to obtain an expression vector, and byusing this expression vector, animals cells such as Chinese hamsterovary (CHO) cells, mouse C127 cells, monkey COS cells and the like aretransformed, and HGF can be obtained from the culture supernatant of thetransformants.

As to HGF thus obtained, there are possibilities that a part of theamino acid sequence will be deleted or substituted with other aminoacid(s), that another amino acid sequence is partially inserted, that 1,2 or more amino acids are attached to the C and/or N terminals, or thatsugars are similarly deleted or substituted. Such HGF analogues aredisclosed in Japanese Patent Kokai No. 130091/1992 and PCT InternationalPublication No. WO90/10651, and they may be also used in the inventionand are included within the scope of the invention.

The above-described HGF can reduce multiple-organ or systemic variousside effects caused by immunosuppressants as shown in Examples mentionedlater. More specifically, nephrotoxicity, hepatoxicity, gastrointestinaldisorder (for example, anorexia, diarrhea or the like), neuropathy (forexample, weakness condition, irritation, convulsion or the like) andinjury of organs are prevented, and recovery of organs is promoted,further, death rate due to side effects can be reduced.

Cells on which HGF acts exist in extremely wide range of organs andtissues such as, for example, liver cell, renal tubular epithelial cell,areolus epithelium, tunica mucosa ventriculi epithelium, blood vesselendothelium, cutis keratinocyte and the like. Therefore, HGF caneffectively reduce multiple-organ or systemic various side effectscaused by immunosuppressants.

A further important point in considering the practical use of HGF asmedicine is that little side effect is recognized if HGF is administeredto animals for long period. It is also a good feature that HGF promotesthe growth of cells only in phase G1, that is, the cells only in thegrowth period, not cells in phase G0, that is, stationary period. Itmeans that it promotes growth and regeneration of injured tissues, butdoes not act at all on intact tissues. Therefore, if HGF is administeredexcessively, or if HGF reaches non-ailing sites through blood or thelike, it does not induce carcinogenic action or excessive growth innormal tissues.

The agent for relieving side effects of the present invention is appliedfor relieving side effects caused by the above-mentioned non-specificimmunosuppressant or specific immunosuppressant in mammals (for example,cow, horse, pig, sheep, dog, cat and the like) including human, andmanifests remarkable effect in reduction of side effects especiallycaused by antibiotic immunosuppressants such cyclosporin, FK506,mitomycin C, daunorubicin and the like. As described above, since actionof HGF reaches wide range of organs and tissues, multiple-organ orsystemic relieving of side effects beyond topical relieving of sideeffects can be accomplished.

The agent for relieving side effects in the invention may be prepared invarious preparation forms (for example, liquid, tablet, capsule), andgenerally it is prepared in the form of injection containing HGF as theactive ingredient alone or together with common carrier, or in the formof oral preparation together with common carrier. The injection may beprepared by the conventional method, and for example, HGF is dissolvedin a proper solvent (for example, sterilized water, buffer solution,physiological saline), filtered and sterilized, and put in a containeraseptically. The content of HGF in the injection may be usually 0.0002to 0.2 w/v%, preferably 0.001 to 0.1 w/v%. As oral preparation, it ismanufactured in various preparation forms, including tablet, granule,fine granule, powder, soft or hard capsule, liquid, emulsion, suspensionor syrup, and these preparations may be manufactured by the conventionalmethod. The HGF content in the preparation may be properly adjusteddepending on the preparation form and the disease to be treated.

In production of the preparation, it is preferable to add a stabilizer,and examples of the stabilizer include albumin, globulin, gelatin,mannitol, glucose, dextran, ethylene glycol and the like. Moreover, thepreparation of the invention may contain other additives necessary forpharmaceutical preparation, such as an excipient, a dissolving aid, anantioxidant, a pain-alleviating agent, an agent for isotonicity and thelike. In liquid preparation, it is preferable to store it under frozenconditions or after the removal of water by a process such asfreeze-drying. The freeze-dried preparation is used by dissolving againin distilled water for injection and the like before use.

The agent for relieving side effects in the invention is administeredthrough various routes depending on the preparation form. For example,the injection is administered by intravenous, intraarterial,subcutaneous, intramuscular and the like. The dose is adjusted properlydepending on symptoms, age and body weight of patient, and generally0.01 mg to 100 mg of HGF is administered once or several times per day.

Industrial Applicability

In the present invention, HGF as the effective component can remarkablyreduce side effects caused by immunosuppressants. Especially, sinceaction of HGF reaches wide range of organs and tissues, multiple-organor systemic relieving of side effects beyond topical relieving of sideeffects can be accomplished. Therefore, according to the presentinvention, restrictions of use and dose of immunosuppressants due toside effects are reduced, success rate of organ transplantation and curerate of various patients to which the immunosuppressants areadministered can be improved and at the same time burden of the patientscan be remarkably reduced.

EXAMPLES

The following Test Examples and Examples further illustrate the presentinvention in detail but are not to be construed to limit the scopethereof.

Here, the human recombinant HGF (hereinafter, referred to as hr-HGF)used in Test Examples was that which was purified from culturesupernatant of CHO cells obtained by transfection of human recombinantHGF cDNA (Nakamura et al., Nature 342, 440-443, 1989; Seki et al.,Biochem. Biophys. Res. Commun. 172, 321-327, 1990).

Test Example 1

Relieving action of HGF against side effect of cyclosporin was testedaccording to the following method.

1. Method

The experimental design of this test is shown in FIG. 1. Acute organdisorder was prepared by daily intraperitoneal administration of 100mg/kg of cyclosporin A (hereinafter, referred to as CsA) to ICR mice.hr-HGF (5 μg for individual per one dose) or a physiological saline ascontrol (0.1 ml) was administered through a tail vein 30 minutes beforethe first CsA administration and then every 12 hours. In FIG. 1, timesfor doses of the above-mentioned medicines are indicated by blacktriangle marks (CsA) and white triangle marks (hr-HGF/physiologicalsaline solution).

Further, mice were sacrificed 60 hours and 108 hours after the first CsAadministration, and were subjected to blood examination, pathologictissue examination and organ regeneration examination. In FIG. 1, timesfor sacrifices are indicated by star marks.

2. Results

(1) Survival rate

The survival rate of test mice is shown in the lower graph of FIG. 1. Inthe control group, 83.3% of mice survived for 3 days and 50% of micesurvived for 5 days. On the contrary, in the hr-HGF-treatment group, allmice survived. Between survival rates of these two groups, there was asignificant difference with 5% significance level in logrank test.

In this way, it was found that administration of hr-HGF remarkablyextended the survival of mice which had acute organ disorder caused byCsA.

(2) Phenomenon and Symptom

Summary of phenomena and symptoms of the hr-HGF administered group andthe control group are shown in Table 1. In the control group, middle tosevere diarrhea was recognized in every example from 1 day after CsAadministration. And, piloerection, hypokinesea and weakness conditionwere simultaneously recognized in most mice following temporaryacrocinesia. After that, mice did not eat food, several mice died withintense convulsion.

On the other hand, in the hr-HGF-treated group, diarrhea was mild ascompared with the control group. And, weakness condition, piloerectionand convulsion were not recognized, and food eating was good and activecondition was maintained.

With respect to body weight change, in the control group, the bodyweight was reduced to 93.6±9.6% of the initial body weight, whereas inthe hr-HGF-treated group, the body weight increased to 102.5±3.0% of theinitial body weight, and there was a significant difference with 5%significance level in unpaired Wilcoxon test.

In this way, it was found that administration of hr-HGF could ease thephenomena and symptoms of side effects by CsA.

                  TABLE 1                                                         ______________________________________                                        (phenomenon and symptom)                                                                                  hr-HGF                                                          Control group administered group                                              3 days   5 days   3 days                                                                              5 days                                  Phenomenon and symptom                                                                      after    after    after after                                   ______________________________________                                        Diarrhea      ++       ++˜+++                                                                           +     +                                       Weakness condition                                                                          +        -˜+                                                                              -     -                                       Piloerection  +        ++       -     -                                       Convulsion    ++       -˜+                                                                              -     -                                       Amount of food to be eaten                                                                  Reduced  Not good Good  Good                                    ______________________________________                                         In the table, -: Normal, +: Slight, ++: Middle, +++: Severe              

(3) Blood Examination

In the control group, slight increases in GPT (Glutamate pyruvatetransaminase) value and total bilirubin value were recognized, and mildhepatic disorder occurred. With respect to blood urea nitrogen value andcreatinine value in blood, there was recognized no statisticallysignificant difference between the values of the control group and thoseof hr-HGF-treated group.

(4) Histopathological Observation

As described above, with respect to the blood urea nitrogen value andcreatinine value in blood, though there was recognized no statisticallysignificant difference between the control group and hr-HGF-treatedgroup, histological difference was recognized. In the control group,weak to middle level vacuolation in wide range in proximal tubules wasrecognized at 3 days after in every mouse, and such change wasrecognized also in three mice which survived to 5 days after.

On the other hand, in the hr-HGF-treated group, though weak vacuolationwas recognized in 4 individuals from 5 individuals at 3 days after, andat 5 days after, kidney was in normal condition except thathistologically slight topical renal tubular necrosis was recognized intwo mice.

Here, middle to severe vacuolation and change in focal cells are shownin Table 2.

As shown in the above description and the result of Table 2, it wasfound that administration of hr-HGF could prevent the organ disordercaused by CsA.

                  TABLE 2                                                         ______________________________________                                        (histopathological observation of proximal tubules)                                                           hr-HGF                                                      Control group     administered group                                          3 days  5 days    3 days                                                                              5 days                                  Histological observation                                                                    after   after     after after                                   ______________________________________                                        Vacuolation*  2/6     2/3       2/5   0/5                                     Change of focal cell*                                                                       4/6     1/3       2/5   3/5                                     ______________________________________                                         *number of animals which occurred middle˜severe change/number of        tested animals                                                           

(5) Organ Regeneration Examination (DNA Synthesis)

To examine whether HGF promoted regenerations of kidney and liver ornot, BrdU (5-bromo-2'-deoxyuridine) was injected into a mouse,subsequently the kidney and liver were stained by a BrdU specificimmunocytochemical method, and DNA syntheses in the kidney and liverwere measured. The results are shown in Table 3.

Ratio of the cells in DNA synthesis period (labeling index) in kidneywas increased to 0.4% from 0.14% in the control group at 3 days after,and to 0.88% in the hr-HGF-treated group.

In liver, the labeling index in the control group was not more than 0.1%at 3 days after, and this value was approximately the same as that of anormal mouse. On the other hand, in the hr-HGF-treated group, itincreased to 0.55%. There was a significant difference with 5%significance level in unpaired Wilcoxon test.

In this way, it was proved that, in the hr-HGF-treated group, increaseof the labeling index was observed, DNA synthesis was activelyconducted, and regenerations of kidney and liver were promoted.

Here, the measurement of the labeling index was conducted by a method ofIshiki et al. (Hepatology 16, 1227-1235, 1992).

                  TABLE 3                                                         ______________________________________                                        (DNA synthesis)                                                                            Labeling index at 3 days after                                                           hr-HGF                                                DNA synthesis                                                                              Control group                                                                            administered group                                    ______________________________________                                        Liver        0.01 ± 0.01 *1                                                                        0.55 ± 0.42 *1                                     Kidney       0.40 ± 0.24 *2                                                                        0.88 ± 0.24 *2                                     ______________________________________                                         *1: p < 0.01 in Wilcoxon test                                                 *2: p < 0.01 in Wilcoxon test                                            

Example 1

A solution containing 1 mg of HGF, 1 g of mannitol and 10 mg ofpolysorbate 80 in 100 ml of physiological saline was asepticallyprepared. 1 ml of the solution was poured into each vial andlyophilized, and then the vial was sealed to obtain a freeze-driedpreparation.

Example 2

A solution containing 1 mg of HGF and 100 mg of human serum albumin in100 ml of 0.02M phosphate buffer (containing 0.15M of NaCl and 0.01% ofpolysorbate 80, pH 7.4) was aseptically prepared. 1 ml of the solutionwas poured into each vial and lyophilized, and then the vial was sealedto obtain a freeze-dried preparation.

Example 3

A solution containing 1 mg of HGF, 2 g of sorbitol, 2 g of glycine and10 mg of polysorbate 80 in 100 ml of distilled water for injection wasaseptically prepared. 1 ml of the solution was poured into each vial andlyophilized, and then the vial was sealed to obtain a freeze-driedpreparation.

What is claimed is:
 1. A method for relieving a side effect caused byimmunosuppressants, which comprises:administering an effective amount ofhepatocyte growth factor (HGF) to reduce said side effect selected fromthe group consisting of hepatic disorder, kidney disorder, neuropathy,gastrointestinal disorder, anorexia, diarrhea, piloerection, fever andconvulsion.
 2. The method of claim 1, wherein the side effect isselected from the group consisting of nephrotoxicity, heptotoxicity,gastrointestinal disorder and neuropathy.
 3. The method of claim 1,wherein the immunosuppressant is an antibiotic immunosuppressant.
 4. Themethod of claim 1, wherein the immunosuppressant is an immunosuppressantwhich blocks intracellular signaling pathways which enhance expressionof a lymphokine gene via activation of a T-cell antigen receptor (TCR)due to antigen stimulation in a T-cell.
 5. The method of claim 1,wherein the immunosuppressant is an immunosuppressant which manifestseffects by binding an immunophilin.
 6. The method of claim 1, whereinthe immunophilin is cyclophilin or FK506 binding protein.